In living cells, prior to cell division, DNA is duplicated by synthesizing a new daughter strand of DNA from the four nucleic acids, cytidine, thymidine, adenosine and guanosine. The daughter strand is a complementary copy of the parent strand of DNA. The period during the cell cycle in which the new DNA strand is synthesized is called the "S-Phase". During DNA synthesis or repair, in any phase, including the S-Phase, cells take up nucleic acids from their environment and insert them into the newly synthesized DNA. Through use of labelled nucleic acids, it is possible to identify those cells which are taking up nucleic acids from the environment and to identify such cells, including S-Phase cells. One of the most commonly used labelled nucleic acids is tritiated Thymidine.
Another known and commonly used label is bromodeoxyuridine (BrdU) which is an analog of the natural nucleic acid thymidine.
Once one of the labelled nucleic acids is incorporated in the cell which is undergoing DNA synthesis, its presence in the cell can be detected in order to identify such cells. Detection of the tritium label can be done with either liquid scintillation counting or by autoradiography, as described by Taylor, et al., Proc. Natl. Acad. Sci., USA 43:122(1957) and R. Baserga and D. Malamud. Autoradiograph: Technique and Application, Harper, New York (1969). Both methods detect the emitted beta radiation of the tritium label.
The process of autoradiography involves overlaying a microscope slide, firstly with cells having the tritium label incorporated in them and secondly with a photographic emulsion. The emulsion-bearing slide is stored for a time sufficient to expose the emulsion to particles emitted from the decaying tritium in the cells; this period usually takes several days and may last as long as several weeks. The slide is photographically developed revealing fogging of the emulsion which is caused by radiation issued from the decaying tritium. This radiation causes the emulsion to be visible over only those cells which have been incorporating the tritiated thymidine into their DNA. The proportion of DNA-synthesizing cells in the total population can then be enumerated by use of a microscope. Approximate quantitation of the amount of radioactive DNA synthesized per cell can be estimated by counting the silver grains from the emulsion overlying each cell.
In the case of liquid scintillation, a population of cells, approximately 10,000, must be counted so that studies of individual cells become impossible. This approach provides information on the average behavior of a group of cells but does not permit single cell analysis.
The process of DNA synthesis can also be determined by the use of halodeoxyuridine (halodu). Since these compounds appear to the cell as thymidine, they are usually incorporated into DNA in place of thymidine. Thus, the synthesis of DNA by the cell can be monitored by measuring in some manner the incorporation of halodeoxyuridine. Recently, a method for the detection of BrdU incorporation into cells which are synthesizing DNA using polyclonal antibodies has been reported; H. G. Gratzner, et al., J. Histochem. Cytochem., 24, 34 (1976); H. G. Gratzner, et al., Res. Comm. Chem. Pathol. Pharmacol., 20, 34 (1978). Also, U.S. Pat. No. 4,529,700 of Gratzner discloses the development of hybridomas that produce monoclonal antibodies to halodu; i.e., to BrdU, Idu, Cldu and Fldu. A monoclonal antibody to halodu can be tagged with a marker which can be detected in a flow cytometry apparatus or by transmission or fluorescent microscopy. The usual marker for flow cytometry is a fluorescent marker.
Unfortunately, all of the methods utilizing antibodies to halodu for the detection of cells synthesizing DNA have relied upon the destruction of the cell membrane and denaturation of double stranded DNA to single stranded DNA prior to the examination of the DNA. The method for denaturation of DNA is usually incubation with a strong inorganic acid, such as hydrochloric acid, or a base, such as sodium hydroxide. This also results in the breaking down of the DNA from double-stranded DNA into single-stranded DNA and destruction of cellular morphology and cellular antigens.
It would be desirable to provide a method for the detection of newly synthesized DNA in cells without exposure of the cells to harsh acidic or basic conditions. Such method would provide a means whereby the newly synthesized DNA can be detected and quantified while at the same time antigenic determinants on the surface of the cell can be detected. This would result in a greatly simplified method for a complete analysis of the cell to determine cell surface markers, such as those recognized by the anti-Leu series of monoclonal antibodies commercially available from the Becton Dickinson Monoclonal Center, Mountain View, Calif., while at the same time detecting and quantifying newly synthesized DNA.
The detection of cells which have newly synthesized DNA as part of their proliferation cycles is highly desirable in the course of monitoring cell growth of normal and tumor cells; the effects of drugs on DNA proliferation during human cancer therapy; improved detection of chemicals which are potentially damaging to DNA to provide a more sensitive method for facilitating the monitoring of human cells for evidence of genetic injury; and as a tool to measure hypoxanthine phosphoribosyl transferase (HPRT) deficiency in human cells.